Membrane-permeable peptide capable of calpain inhibition

ABSTRACT

5-mer peptides that can inhibit calpain I and calpain II are disclosed. The peptide sequence is as follows: Leu or Ala)-(Xaa)-(Asp or Glu)-(Xaa)-(Leu or Met), where Xaa can be any amino acid.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to provisional application60/438,318, filed Jan. 7, 2003.

FIELD OF THE INVENTION

[0002] The invention pertains to peptides that are membrane-permeableand effective at protecting neurons against calpain-mediated cell death.In particular, the peptides of the invention are similar to domains Aand C of calpastatin, which is a known inhibitor of calpain I andcalpain II. However, unlike calpastatin, the peptides of the inventiondo not target an active cysteine of calpain.

BACKGROUND OF THE INVENTION

[0003] Calpain is a cytosolic calcium-dependent cysteine protease. It isfound in all mammalian tissue and cell types. The calpain family ofproteases consists of two recognized proteins.

[0004] The first member of the calpain family is calpain I or μ-calpainwhich is the high sensitivity form and is activated by a low calciumconcentration (2-75 μM). Calpain I is concentrated in synapses andneuronal cell bodies.

[0005] The second member of the calpain family is calpain II orm-calpain. It has a ribbon like structure and is the lower sensitivityform. It is activated by higher concentrations of calcium (200-800 μM).Calpain II is the dominant form.

[0006] Calpastatin is a known endogenous inhibitor of calpain I andcalpain II. It has four internally repeated domains, each of whichindependently binds a calpain molecule in its active, Ca⁺⁺-boundconformation with high affinity.

[0007] Calpains play an important role in various physiologicalprocesses. These processes include, inter alia, the cleavage ofregulatory proteins such as protein kinase C (PKC) and degradation ofcytoskeletal or microtubule-associated proteins (MAP) such as tau.Research has demonstrated that calpain inhibitors have improved recoveryfrom the memory performance deficits and neuromotor disturbances.Calpain inhibitors inhibit the release of the 13-AP4 protein. Therefore,it has been suggested that they have a potential use as therapeuticagents in Alzheimer's disease. Calpain inhibitors have also had aprotective effect on hypoxia-damaged kidneys and have had favorableeffects following cardiac damage produced by ischemias of the heart(e.g., myocardial infarction) or reperfusion. It has also been foundthat calpain inhibitors have cytotoxic effects on tumor cells. Elevatedcalpain levels are also implicated in the pathophysiology of cerebralischemia, platelet activation, NF-KB activation, muscular dystrophy,cataract progression and rheumatoid arthritis. with variouspathophysiological processes such as, inflammations, musculardystrophies, cataracts of the eyes, and injuries to the central nervoussystem (e.g., trauma).

[0008] Calpains reside in the cytosol of cells and are activated by Ca⁺⁺at a physiological pH. Its proteolytic activity appears to be selectiveagainst certain target proteins, such as components of the cytoskeletonand calmodulin-dependent enzymes.

[0009] Excessive excitation by a neurotransmitter glutamate can lead todeath of nerve cells (neurons) and neurodegeneration. It is believedthat toxic effects of glutamate comes from overactivation of its targetglutamate receptors (e.g., under ischemic conditions or stroke). This inturn produces an influx of calcium ion (Ca⁺⁺) into the neurons. The riseof cellular Ca⁺⁺ level triggers the activation of calpain I. Calpain Ithen goes on to degrade cytoskeletal protein such as spectrin, which isbelieved to disrupt normal cellular functions, and eventually leads tocell death.

SUMMARY OF THE INVENTION

[0010] The invention is directed to proteins and methods of using theproteins that inhibit calpains I and II. The method includes contactingthe cell with an effective amount of the protein for inhibiting calpainsI and II.

[0011] In one aspect of the invention there is provided a peptidecomprising an amino acid sequence (Leu or Ala)-(Xaa)-(Asp orGlu)-(Xaa)-(Leu or Met), where Xaa is any amino acid. In a preferredembodiment the peptide has the amino acid sequence, Leu-Ser-Glu-Ala-Leu(LSEAL).

[0012] In a another aspect of the invention there is provided a methodfor inhibiting calpain comprising contacting a cell with an amount of apeptide comprising the amino acid sequence (Leu or Ala)-(Xaa)-(Asp orGlu)-(Xaa)-(Leu or Met), where Xaa is any amino acid.

[0013] In yet another aspect of the invention there is provided a methodfor inhibiting calpain-dependent cell death comprising contacting a cellwith an effective amount of a cell membrane permeable peptide comprisingthe sequence (Leu or Ala)-(Xaa)-Asp or Glu)-(Xaa)-(Leu or Met), whereXaa is any amino acid.

[0014] The invention also provides a method for protecting cells from UVirradiation-induced cell death comprising exposing the cells to aneffective amount of a polypeptide having the amino acid sequence, (Leuor Ala)-(Xaa)-Asp or Glu)-(Xaa)-(Leu or Met), where Xaa is any aminoacid.

[0015] Other objects, features and advantages of this invention willbecome apparent upon reading the following detailed description andreferring to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the same becomebetter understood by reference to the following detailed descriptionwhen considered in connection with the accompanying drawings.

[0017]FIG. 1 is a graphical illustration of the relationship of doseresponse of Leu-Ser-Glu-Ala-Leu (LSEAL) (SEQ ID NO. 1) with ionomycin(ionomycin toxicity) with respect to cell death.

[0018]FIG. 2 is a plot of the relationship of ultraviolet light (UV)exposure and cell death between cultured Samples of cortical neuronscontaining no LSEAL and Samples containing LSEAL.

[0019]FIG. 3 is a plot of the relationship of the effect of LSEAL on taudegradation by calpain I.

DETAILED DESCRIPTION OF THE INVENTION

[0020] This invention relates to 5-mer peptides and methods of usingthese peptides for inhibiting calpain. In particlular, the peptidescomprise the following sequence: (Leu or Ala)-(Xaa)-Asp orGlu)-(Xaa)-(Leu or Met), where Xaa can be any amino acid (SEQ ID NO. 2).

[0021] In one embodiment, Xaa is selected from the group consisting ofAla, Arg, Ile, Leu, Pro, Phe, Ser, Thr and Tyr. In another embodiment,the peptides include Leu-Ser-Glu-Ala-Leu (SEQ ID NO. 3),Leu-Ala-Asp-Ala-Leu (SEQ ID NO. 4), Leu-Phe-Glu-Ala-Leu (SEQ ID NO. 5),Leu-Tyr-Asp-Ala-Leu (SEQ ID NO. 6), Leu-Leu-Glu-Ala-Leu (SEQ ID NO. 7),Leu-Arg-Asp-Ala-Leu (SEQ ID NO. 8), Leu-Tyr-Asp-Ala-Met (SEQ ID NO. 9),Leu-Thr-Asp-Ala-Leu (SEQ ID NO. 10), Ala-Leu-Asp-Ala-Leu (SEQ ID NO.11), Leu-Leu-Asp-Ala-Leu (SEQ ID NO. 12), Pro-Ile-Asp-Ala-Leu (SEQ IDNO. 13) Leu-Ile-Asp-Glu-Leu (SEQ ID NO. 14), Ala-Ile-Asp-Ala-Leu (SEQ IDNO. 15), Leu-Ile-Asp-Thr-Leu (SEQ ID NO. 16), Leu-Ala-Asp-Ser-Leu (SEQID NO. 17), and Leu-Ser-Asp-Ser-Leu (SEQ ID NO. 18). All of these aminoacid sequences are similar to the binding region of the endogenousinhibitor calpastatin.

[0022] One particular five amino acid peptide comprisingLeu-Ser-Glu-Ala-Leu (SEQ ID NO. 1) (hereinafter LSEAL is particularlypreferred for its ability to inhibit calpain activity both in vitro andin situ. It was found that this peptide is membrane-permeable andeffective at protecting neurons against calpain-mediated cell death intwo different paradigms.

[0023] LSEAL is similar to domains A and C of calpastatin which havebeen extensively studied by others in which they have shown that regionsof calpastatin that are similar to our peptides are required for fullinhibition of calpains. In addition, other groups have shown thatmutations of specific amino acids in domains A or C, which are theequivalent amino acids in our peptide (SEQ ID NO. 1) of the presentinvention, result in reduced calpastatin inhibitory action, indicatingthat the sequence is likely optimal for calpain inhibition. Thesefindings suggest that the peptides of the invention can mimic theactions of calpastatin.

[0024] The LSEAL peptide inhibits calpain activity in vitro towards thesubstrate tau protein. Also, this peptide prevents calpain-dependentcell death in two model systems of low-dose ionomycin treatment as wellas UV-induced cell toxicity. While other synthetic inhibitors areavailable for calpains, this peptide has several advantages.

[0025] First, all other calpain inhibitors target the active sitecysteine of calpain. This leads to significant specificity loss as manyother proteases have cysteine residues at their active sites that havebeen shown to be modified by these other inhibitors. As such, it isextremely unlikely that the peptides will react with other proteases.The peptides of the invention mimics a key portion of the calpastatinmolecule required for its endogenous and specific inhibitory action tothe calpains.

[0026] Second, the peptides of the invention are membrane-permeablewithout the need for other modifications. The peptides of the presentinvention can affect changes inside a cell, and are likely to producemore specific effects due to calpain, and are more membrane-permeablethan all previous calpain inhibitors.

[0027] The description below sets forth the method used to identify thepeptide(s) using the Ph.D-12™ phage display system from New EnglandBiolabs (NEB). The protocol for determining the number of phages is fromthe NEB Ph.D-12™ Kit manual, verson 2.5. In this method, the active-siteof calpain I was irreversibly inactivated or blocked with CalpainInhibitor IV, and the peptide(s) were isolated as calcium-dependentbinding peptides. Therefore, these peptides have a calpain Iconformation-dependent binding sequence.

[0028] A mixture of 500 μL of Hepes balanced salt solution (HBSS) (2×concentration) was mixed with 475 μL distilled water, 2 μL of calpain I,0.5 μL of calpain inhibitor IV and 5 μL of 200 mM CaCl₂. Calpaininhibitor IV was in a buffer containing 20 mM Hepes, 0.7 mM Na₂HPO₄, 137mM NaCl, 5 mM KCl, 6 mM glucose at a pH 7.4 (1×HBSS) in the presence of1 mM CaCl₂. The calpain inhibitor IV was added to permanently block theactive cysteine of calpain I. CaCl₂ was added to activate the calpain Iactive site. A single well of a Nunc-Immuno™ microwell plate with aMaxiSorp™ surface was filled with 200 μL of the mixture and incubated atroom temperature for 15 minutes. The plate was covered and shaken in anorbital shaker for 30 minutes at 37° C. and at 250 rpm.

[0029] The well was rinsed three times with a rinse solution comprising1× concentration of HBSS and 1 mM CaCl₂ to remove excess and/ornon-bound calpain I. To prevent direct binding of the phage to theSample well, the well and a second well (hereinafter a control well)were treated with a blocking agent, a 3% bovine serum albumin (BSA) in1×HBSS in 1 mM CaCl₂. The plate was then covered and shaken in theorbital shaker for 30 minutes at 37° C. and at 250 rpm. The control welland the Sample well were then rinsed three times with the rinsesolution.

[0030] The phage (approximately 2×10¹¹ cells) were added to the controlwell. The plate was covered and shaken in the orbital shaker for 30minutes at 37° C. and at 250 rpm. The phage were then transferred to theSample well containing calpain I and the plate was covered and shaken inthe orbital shaker for 30 minutes at 37° C. and at 250 rpm. Afterincubation, the plate was washed 50 times with the rinse solution toremove excess or non-bound phage. The final wash, i.e., the 50th wash,was saved and placed in a microtube.

[0031] In accordance with the protocol in the NEB instruction manual,the Sample well was eluted four times with HBSS and 1 mMethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),a calcium chelating agent. Each elution with EGTA was for a period of 10to 15 minutes at room temperature and 60 rpm shaking. Each of the fourelutions were saved. The well was then eluted with a solution containing0.2 M glycine having a pH of 2.2, and then with 4M solution of urea. Asfor the elution with glycine, this step was performed for 5 minutes atroom temperature followed by a neutralization step by adding 150 μL of1M tris-HCl to raise the pH to 9.1 to prevent killing of the phage. Theglycine and urea elutants were combined. Each elutant, including thesaved final wash was titered to measure the number of phage remaining inthe elutant.

[0032] Amplified phage were then titred to determine their concentrationand the process was repeated four times using the amplified phage fromthe previous round. The phage from the final screening process weresequenced to determine a consensus binding sequence. The titre resultswere as follows:

[0033] Round 1 (×10³): Final Wash=100; Elution 1=800; Elution 2=615;Elution 3=411; Elution 4=362; Glycine/Urea combination=100.

[0034] Round 2 (×10²): Final Wash=2; Elution 1=1400; Elution 3=120;Elution 4=38; Glycine/Urea=75.

[0035] Round 3 (×10³): Final Wash=7; Elution 1=14,130; Elution 2=5495;Elution 3=1800; Elution 4=1570; Glycine/Urea=3140.

[0036] Round 4 was not titred.

[0037] In order to demonstrate the effectiveness of the inhibition ofcalpain by the peptides of this invention, two in situ experiments wereconducted using cultured cortical neurons from rat brains to show thatthe LSEAL peptide inhibited cell death. In the first experiment, celldeath of the neurons was induced by exposing the culture to ionomycin.In the second experiment, cell death was induced by exposing the neuronsto 30 and 60 mJ/cm² of UV light. The exposure to either ionomycin or UVlight caused calpain I to be activated because of ionomycin-mediatedcalcium flux or through UV-induced receptors, channels or pore-likestructures. Calcein acetoxymethyl ester was used as indicator of liveneuron cells while propidium iodide was used an indicator of dead neuroncells.

[0038] In the first experiment, ten Samples were prepared. Sample 1 wasa control Sample and included the cultured cortical neurons only. Abaseline for the average percentage of cell death was determined to be19%. In Sample 2, 0.4 μM ionomycin was added to the cultured neuroncells to activate calpain I. No LSEAL was added to these Samples. Theaddition of ionomycin to these Samples caused the percentage of deadcells to more than triple to an average of 68%. Sample 3 was the same asSample 2 except that 0.00001 micromolar of LSEAL was added before theionomycin was added. The percentage of dead cells averaged of 106% of noLSEAL. Sample 4 was the same as Sample 3 except that 0.0001 micromolarof LSEAL was added. The percentage of cell death was 83% of no LSEAL.Sample 5 was the same as Sample 4 except that 0.001 micromolar LSEAL wasadded. The percentage of cell death was 63% of no LSEAL. Sample 6 wasthe same as Sample 5 except that 0.01 micromolar LSEAL was added. Thepercentage of cell death was 61% of no LSEAL. Sample 7 was the same asSample 6 except that 0.1 micromolar LSEAL was added. The percentage ofcell death was 57% of no LSEAL. Sample 8 was the same as Sample 7 exceptthat 1.0 micromolar LSEAL was added. The percentage of cell death was48% of no LSEAL. Sample 9 was the same as Sample 8 except that 10.0micromolar LSEAL was added. The percentage of cell death was 35% of noLSEAL. Sample 10 was the same as Sample 9 except that 100 micromolarLSEAL was added. The percentage of cell death was 33% of no LSEAL. It isclear from this experiment that the peptide of the invention inhibitedcalpain I activity. The results are summarized in Table 1 below and theresults are graphically illustrated in FIG. 1 TABLE 1 Percentage Averagecell death Number (ratioed to 0 Standard Error of Samples MicromolarLSEAL) of the Mean (n =) 0 LSEAL 100 4.8 13 (Sample 2) .00001 106 13.013 LSEAL (Sample 3) .0001 83 10.7 3 LSEAL (Sample 4) .001 LSEAL 63 14.24 (Sample 5) 0.01 LSEAL 61 9.4 5 (Sample 6) 0.1 LSEAL 57 5.9 7 (Sample7) 1 LSEAL 48 4.9 4 (Sample 8) 10 LSEAL 35 3.3 6 (Sample 9) 100 LSEAL 338.0 3 (Sample 10) Cell death percentages raw data: Percent (raw data)Control 19 (vehicle treatment only) - Sample 1 Ionomycin 68 alone -Sample 2

[0039] In the second experiment, cell death of the cultured corticalneurons was induced by UV light. Samples 11, 12 and 13 were controlSamples which did not contain any LSEAL. Sample 11 shows the normal %dead cells with no exposure to UV light. The percentage of cell deathwas 18%. Sample 14 is the same as Sample 11, except for the addition of100 μM LSEAL to the culture. The percentage of cell death was reducedfrom 18% to 14%. Sample 12 was exposed to 30 mJ/cm² of UV light. Thepercentage of cell death was 58%. The inclusion of the LSEAL beforeexposure reduced the percentage of cell death to 25%. See Sample 15. Ifthe exposure is increased to 60 mJ/cm², the cell death percentage is 74%for the culture which does not contain LSEAL (see Sample 13), and 30%for a culture containing LSEAL (see Sample 16). The data clearly showthat the peptide of the present invention protects neurons fromUV-induced, calpain-dependent cell death. The data for the experiment isset forth below in Tables 3 and 4, and is graphically illustrated inFIG. 2. TABLE 2 Sample Live Cells Dead Cells Total Cells % Dead Cells 1136 7 43 18 12 25 33 58 58 13 17 42 59 74 14 30 5 35 14 15 78 26 104 2516 56 25 81 30

[0040] TABLE 3 UV Light Live Dead Total % Dead Std Exposure SamplesCells Cells Cells Cells Dev  0 mJ/cm² 11 (Control) 36 7 43 18 10 14 30 535 14 10 30 mJ/cm² 12 (Control) 25 33 58 58 1 15 78 26 104 25 2 60mJ/cm² 13 (Control) 17 42 59 74 17 16 56 25 81 30 6

[0041] Another experiment was conducted to show the effect of theproteins of the present invention on the tau protein. The tau protein isa microtubule-associated protein (MAP). Tau is a well-described calpainI substrate. The addition of 100 μM LSEAL inhibits degradation of theprotein. The results are shown in FIG. 3.

[0042] While the salient features have been illustrated and describedwith respect to particular embodiments, it should be readily apparentthat modifications can be made within the spirit and scope of theinvention, and it is therefore not desired to limit the invention to theexact details shown and described.

1 18 1 5 PRT Homo sapiens 1 Leu Ser Glu Ala Leu 1 5 2 5 PRT Homo sapiensMISC_FEATURE (1)..(1) X is L or A 2 Xaa Xaa Xaa Xaa Xaa 1 5 3 5 PRT Homosapiens 3 Leu Ser Glu Ala Leu 1 5 4 5 PRT Homo sapiens 4 Leu Ala Asp AlaLeu 1 5 5 5 PRT Homo sapiens 5 Leu Phe Glu Ala Leu 1 5 6 5 PRT Homosapiens 6 Leu Tyr Asp Ala Leu 1 5 7 5 PRT Homo sapiens 7 Leu Leu Glu AlaLeu 1 5 8 5 PRT Homo sapiens 8 Leu Arg Asp Ala Leu 1 5 9 5 PRT Homosapiens 9 Leu Tyr Asp Ala Met 1 5 10 5 PRT Homo sapiens 10 Leu Thr AspAla Leu 1 5 11 5 PRT Homo sapiens 11 Ala Leu Asp Ala Leu 1 5 12 5 PRTHomo sapiens 12 Leu Leu Asp Ala Leu 1 5 13 5 PRT Homo sapiens 13 Pro IleAsp Ala Leu 1 5 14 5 PRT Homo sapiens 14 Leu Ile Asp Glu Leu 1 5 15 5PRT Homo sapiens 15 Ala Ile Asp Ala Leu 1 5 16 5 PRT Homo sapiens 16 LeuIle Asp Thr Leu 1 5 17 5 PRT Homo sapiens 17 Leu Ala Asp Ser Leu 1 5 185 PRT Homo sapiens 18 Leu Ser Asp Ser Leu 1 5

What is claimed is:
 1. A cell membrane permeable peptide comprising asequence (Leu or Ala)-(Xaa)-(Asp or Glu)-(Xaa)-(Leu or Met), where Xaais any amino acid.
 2. The peptide of claim 1 wherein Xaa is an aminoacid selected from the group consisting of Ala, Arg, Ile, Leu, Pro, Phe,Ser, Thr and Tyr.
 3. The peptide of claim 1, wherein the sequencedefines a peptide selected from the group consisting ofLeu-Ser-Glu-Ala-Leu, Leu-Ala-Asp-Ala-Leu, Leu-Phe-Glu-Ala-Leu,Leu-Tyr-Asp-Ala-Leu, Leu-Leu-Glu-Ala-Leu, Leu-Arg-Asp-Ala-Leu,Leu-Tyr-Asp-Ala-Met, Leu-Thr-Asp-Ala-Leu, Ala-Leu-Asp-Ala-Leu,Leu-Leu-Asp-Ala-Leu, Pro-Ile-Asp-Ala-Leu, Leu-Ile-Asp-Glu-Leu,Ala-Ile-Asp-Ala-Leu, Leu-Ile-Asp-Thr-Leu, Leu-Ala-Asp-Ser-Leu, andLeu-Ser-Asp-Ser-Leu.
 4. The peptide of claim 1, wherein the peptide isLeu-Ser-Glu-Ala-Leu.
 5. An isolated polynucleotide encoding the peptideof claim
 1. 6. An isolated polynucleotide encoding the polypeptide ofclaim
 5. 7. An isolated polypeptide encoding a combination of any of thepeptides of claim
 3. 8. A method of inhibiting calpain comprising thestep of contacting a cell with an effective amount of a cell membranepermeable peptide comprising the sequence (Leu or Ala)-(Xaa)-(Asp orGlu)-(Xaa)-(Leu or Met), where Xaa is any amino acid.
 9. The method ofclaim 8 wherein Xaa is selected from the group consisting of Ala, Arg,Ile, Leu, Pro, Phe, Ser, Thr and Tyr.
 10. The method of claim 8, whereinthe sequence defines a peptide is selected from the group consisting ofLeu-Ser-Glu-Ala-Leu, Leu-Ala-Asp-Ala-Leu, Leu-Phe-Glu-Ala-Leu,Leu-Tyr-Asp-Ala-Leu, Leu-Leu-Glu-Ala-Leu, Leu-Arg-Asp-Ala-Leu,Leu-Tyr-Asp-Ala-Met, Leu-Thr-Asp-Ala-Leu, Ala-Leu-Asp-Ala-Leu,Leu-Leu-Asp-Ala-Leu, Pro-Ile-Asp-Ala-Leu, Leu-Ile-Asp-Glu-Leu,Ala-Ile-Asp-Ala-Leu, Leu-Ile-Asp-Thr-Leu, Leu-Ala-Asp-Ser-Leu, andLeu-Ser-Asp-Ser-Leu.
 11. The method of claim 8, wherein the peptide isLeu-Ser-Glu-Ala-Leu.
 12. A method of inhibiting calpain-dependent celldeath comprising contacting a cell with an effective amount of a cellmembrane permeable peptide comprising the sequence (Leu orAla)-(Xaa)-(Asp or Glu)-(Xaa)-(Leu or Met), where Xaa is any amino acid.13. The method of claim 12 wherein Xaa is an amino acid selected fromthe group consisting of Ala, Arg, Ile, Leu, Pro, Phe, Ser, Thr and Tyr.14. The method of claim 12, wherein the peptide is selected from thegroup consisting of Leu-Ser-Glu-Ala-Leu, Leu-Ala-Asp-Ala-Leu,Leu-Phe-Glu-Ala-Leu, Leu-Tyr-Asp-Ala-Leu, Leu-Leu-Glu-Ala-Leu,Leu-Arg-Asp-Ala-Leu, Leu-Tyr-Asp-Ala-Met, Leu-Thr-Asp-Ala-Leu,Ala-Leu-Asp-Ala-Leu, Leu-Leu-Asp-Ala-Leu, Pro-Ile-Asp-Ala-Leu,Leu-Ile-Asp-Glu-Leu, Ala-Ile-Asp-Ala-Leu, Leu-Ile-Asp-Thr-Leu,Leu-Ala-Asp-Ser-Leu, and Leu-Ser-Asp-Ser-Leu.
 15. The method of claim 12wherein the peptide is Leu-Ser-Glu-Ala-Leu.
 16. A method for protectingcells from UV irradiation-induced cell death comprising exposing thecells to an effective amount of a polypeptide having the amino acidsequence, (Leu or Ala)-(Xaa)-(Asp or Glu)-(Xaa)-(Leu or Met), where Xaais any amino acid.
 17. The method of claim 16 wherein Xaa is an aminoacid selected from the group consisting of Ala, Arg, Ile, Leu, Pro, Phe,Ser, Thr and Tyr.
 18. The method of claim 16, wherein the sequencedefines a peptide selected from the group consisting ofLeu-Ser-Glu-Ala-Leu, Leu-Ala-Asp-Ala-Leu, Leu-Phe-Glu-Ala-Leu,Leu-Tyr-Asp-Ala-Leu, Leu-Leu-Glu-Ala-Leu, Leu-Arg-Asp-Ala-Leu,Leu-Tyr-Asp-Ala-Met, Leu-Thr-Asp-Ala-Leu, Ala-Leu-Asp-Ala-Leu,Leu-Leu-Asp-Ala-Leu, Pro-Ile-Asp-Ala-Leu, Leu-Ile-Asp-Glu-Leu,Ala-Ile-Asp-Ala-Leu, Leu-Ile-Asp-Thr-Leu, Leu-Ala-Asp-Ser-Leu, andLeu-Ser-Asp-Ser-Leu.
 19. The method of claim 16, wherein the peptide isLeu-Ser-Glu-Ala-Leu